Method for the treatment of inflammatory disorders

ABSTRACT

A method for the treatment of inflammatory disorders is disclosed, particularly the treatment of arthritis. The method comprises the administration of a function blocking antibody which is capable of binding an epitope of VLA-1.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/108,581, filed Apr. 18, 2005, which is a continuation of U.S.application Ser. No. 09/996,738, filed Nov. 30, 2001, now U.S. Pat. No.6,955,810, which is a continuation of PCT/US00/15004, filed Jun. 1,2000, which claims priority from U.S. Provisional Application No.60/185,336 filed Feb. 29, 2000, and U.S. Provisional Application No.60/137,038 filed Jun. 1, 1999.

BACKGROUND OF THE INVENTION

Integrins are cell surface protein complexes that form a large class ofcell-surface molecules mediating adhesion of cells to each other andtheir surrounding. Cells need to adhere to each other and to othermolecules in their environment in many developmental and physiologicalprocesses. Examples include the creation of tissues and organs and themaintenance of their integrity. Including amongst these physiologicalprocesses are inflammatory disorders.

One of the key steps during the inflammatory process involves theextravasation of cells out of the blood vessels, into the tissues, andtowards the site of infection. The role of adhesion molecules in thisprocess is often broken down into a three step model involving initialleukocyte ‘rolling’ on inflamed endothelium, followed by firmattachment, and resulting in transendothelial migration of leukocytesinto the inflamed tissues (Hynes, R. O. 1992 Cell 69:11-25; Springer, T.A. 1992 Cell 76:301-314). A further critical step in the inflammatorycascade, and one that has not been extensively explored, occurs withinthe peripheral tissues where infiltrating, as well as resident cells,need to migrate towards the site of infection, recognize foreignantigen, and undergo cellular activation in order to perform theireffector functions. To directly assess the importance in inflammation ofinterstitial adhesive interactions in isolation from the role adhesiveinteractions play in leukocyte recruitment, we have focused on theimportance of adhesion molecules of the integrin family and fragmentsthereof, and their role in animal models of inflammation, particularlyarthritis.

SUMMARY OF THE INVENTION

The present invention provides a method for treatment of inflammatorydisorders in a subject. Specifically, the invention provides a methodfor treatment of arthritis.

More particularly, the invention provides a method for the treatment ofan inflammatory disorder in a subject comprising administering to thesubject a pharmaceutical composition comprising an effective amount ofan α1β1 function blocking antibody or a fragment of the antibody,wherein the α1β1 function blocking antibody or fragment is capable ofbinding an epitope of VLA-1 comprising amino acid residues 91-96,Val-Gln-Arg-Gly-Gly-Arg.

The anti-integrin antibody can be selected from the group consisting ofa human antibody, a chimeric antibody, a humanized antibody andfragments thereof. The anti-integrin antibody can be a monoclonal orpolyclonal antibody.

The invention further provides a method for treating inflammatorydisorders in a subject that is a human or animal subject.

All of the cited literature in the preceding section, as well as thecited literature included in the following disclosure, are herebyincorporated by reference.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Collagen-binding integrins α1β1 and α2β1 on activatedleukocytes. FIG. 1A is a graph depicting flow cytometric analysis of α1and α2β1 integrin expression on IL-2-activated splenocytes (d 11). Cellswere labeled with either anti-α1 mAb, anti-α2 mAb, or non-bindingcontrol mAb (grey lines), and followed by FITC-anti-hamsterimmunoglobulin. FIG. 1B is a bar graph depicting the effect of anti-α1and anti-α2 mAbs on leukocyte adhesion to collagen. 10⁵ IL-2 activatedsplenocytes were treated with indicated mAbs for 15 min before platingonto either type IV or type I collagen-coated wells for 1 h at 37° C.Adhesion was calculated as illustrated in Example 1, and expressed as %adhesion relative to control mAb-treated cells. Adhesion assays weredone in triplicate, and at least three independent experiments wereperformed. One representative experiment is shown.

FIG. 2. Effect of anti-integrin mAbs on the effector phase ofdelayed-type hypersensitivity. SRBC-sensitized mice were injected i.p.with the indicated mAbs 1 h prior to SRBC challenge. Footpad thicknesswas measured 20 h after antigen challenge, and results shown as %increase in footpad thickness±SEM as illustrated in Example 2. Thesedata represent a summary of eight experiments with n=79 (PBS), 68(control hamster Ig), 68 (anti-α1), 29 (anti-α2), 18 (anti-α1+anti-α2),45 (anti-α4), 18 (anti-α5), 20 (anti-α6), and 10 (anti-β1). The mAbsused were: Ha4/8 (control hamster Ig group 2), Ha31/8 (anti-α1), Ha1/29(anti-α2), PS/2 (anti-α4), 5H10-27 (anti-α5), GoH3 (anti-α6), and HMβ3-1(anti-β1).

FIG. 3. Effect of anti-integrin mAbs on the effector phase of contacthypersensitivity. FITC-sensitized mice were injected i.p. with theindicated mAbs 4 h prior to FITC challenge. Ear thickness was measuredat baseline and 24 h later, and results shown as % increase in earthickness±SEM as illustrated in Example 3. These data represent asummary of nine experiments with n=74 (PBS), 60 (control hamster Ig), 26(anti-ICAM-1), 44 (anti-α1), 44 (anti-α2), 38 (anti-α1+anti-α2), 36(anti-α4), 16 (anti-α5), 26 (anti-α4+anti-α5), 24 (anti-α6), and 22(anti-β1). The hamster mAbs used were: Ha4/8 (control hamster Ig group2), Ha31/8 (anti-α1), Ha1/29 (anti-α2), HMβ1-1 (anti-β1), 3E2(anti-ICAM-1); the rat mAbs used were: R35-95 and R35-38 (control ratIgG2a and rat IgG2b, respectively), PS/2 (anti-α4), 5H10-27 (anti-α5),GoH3 (anti-α6).

FIG. 4. Contact hypersensitivity responses in α1-deficient mice comparedto wild-type mice. FITC-sensitized mice were injected i.p. withindicated mAbs 4 h prior to FITC challenge. Ear thickness was measuredat baseline and 24 h later, and results shown as % increase in earthickness±SEM as illustrated in Example 4. Groups of four to five miceper condition were used, and all experiments were performed a minimum ofthree times. One representative experiment is shown.

FIG. 5. Effect of anti-α1 and anti-α2 mAbs on croton oil-inducednon-specific inflammation. Mice were injected i.p. with indicated mAbs 4h prior to ear painting with croton oil. Ear thickness was measured atbaseline and 24 h later, and results shown as % increase in earthickness±SEM as illustrated in Example 5. Groups of four to five miceper condition were used, and all experiments were performed a minimum ofthree times. One representative experiment is shown.

FIG. 6. Effect of anti-α1 and α2 mAbs in collagen mAb-induced arthritis.Mice were injected i.p. with anti-collagen mAbs at d 0, followed by LPSon day 3. Mice were injected i.p. with indicated mAbs every 3^(rd) daystarting on d 0. Clinical arthritis was apparent 2-3 d following LPSinjection and continued for several weeks. Each limb was evaluated on a0 to 4 scale every 3^(rd) day as illustrated in Example 6 and resultsare expressed as the mean arthritic score between d 9 and d 15 (±SEM) ofall four limbs. These data represent a summary of four experiments witheach experiment consisting of groups of three to four mice percondition.

FIG. 7. Administration of anti-α1 or anti-α2 mAbs inhibits leukocyteinfiltration into footpads during a DTH response. The experiment wasperformed as described in FIG. 2. Footpads were excised 20 h followingantigen challenge and tissue sections stained with hematoxylin andeosin. Tissue sections are from footpads of either unchallenged mice (A)or SRBC-sensitized mice challenged with SRBC (B-H). Mice were treated 1h prior to challenge with either PBS (B), control hamster Ig (C, G),anti-α1 (D), anti-α2 (E) or a combination of anti-α1 and anti-α2 mAbs(F, H). Magnification: ×100 (A-F), ×400 (G-H).

FIG. 8. α1β1 is expressed on infiltrating leukocytes in footpads duringa DTH response. Immunohistochemical staining of infiltrating leukocytesfrom an untreated inflamed footpad 20 h after antigen challenge. FIG. 8Aare images of serial sections stained directly with Alexa488-conjugatedcontrol mAb and anti-α1 mAb. FIG. 8A is a panel of images of dualimmunofluorescent staining with Alexa488-conjugated anti-α1 mAb andPhycoerythrin (PE)-conjugated cell lineage-specific mAbs. PE-conjugatedmAbs utilized were specific for granulocytes/monocytes (anti-CD11b),neutrophils (anti-Ly6G/Gr-1), and T lymphocytes (anti-CD3).Magnification: ×400.

FIG. 9. Effect of anti-α1 and α2 mAbs in collagen mAb-induced arthritis.(A). Preventative treatment of mice with either anti-α1 or anti-α2 mAbdecreases arthritic score. Mice were treated with anti-collagen mAbs atd 0, followed by LPS on d 3. Arthritis was apparent by d 6 and continuedfor several weeks. Mice were treated with the indicated mAbs every3^(rd) day starting on d 0. Each limb was evaluated and scored on a 0 to4 scale every 3^(rd) day. Results are expressed as the mean arthriticscore between d 9 and d 15 (±SEM) of all four limbs (maximum score of16). Groups of 4 mice per condition were used; the average of 12experiments is shown. (B). α1-deficient mice have a reduced arthriticscore comparable to anti-α1 mAb-treated wild-type mice. Experimentaldetails and scoring are as outlined above. Groups of 4 mice percondition were used; the average of 2 experiments is shown.

FIG. 10. Effect of anti-α1 mAb treatment on the immunopathology ofarthritic joints. Anti-α1 mAb treatment reduces leukocytic infiltration,adherence of cells to joint surfaces, and cartilage destruction asevidenced by proteoglycan loss. Hind limbs from normal mice (A-D) orarthritic mice (d 8) receiving either control hamster Ig (E-H) oranti-α1 mAb treatment. (I-L). Limbs were photographed (A, E, I),excised, and tissue sections stained either with hematoxylin/eosin (B-C,F-G, J-K) or with toluidine blue to detect proteoglycan (D, H, L).Magnification: ×16 (B, F, J); ×160 (C, G, K); ×200 (D, H, L).

FIG. 11. Development of arthritis is delayed in the absence oflymphocytes and inhibition of arthritis by anti-α1 mAb occurs in theabsence of lymphocytes. Wild-type B6,129 or RAG-1-deficient B6,129 micewere treated with anti-collagen mAbs at day 0, followed by LPS on day 3.Arthritis was apparent by day 6 and continued for several weeks. Micewere treated with the indicated mAbs every 3^(rd) day starting on day 0.Each limb was evaluated and scored on a 0 to 4 scale every 3^(rd) day.Results are expressed as the mean arthritic score per limb (maximumscore of 4). Groups of 4 mice per condition were used.

FIG. 12. Dose response of anti-α1 mAb inhibition of arthritis. Wild-typeBalb/c mice were treated with anti-collagen mAbs at day 0, followed byLPS on day 3. Arthritis was apparent by day 6 and continued for severalweeks. Mice were treated i.p. with the indicated dose of either Ha4/8(isotype control) or Ha31/8 (anti-α1) mAbs every 3^(rd) day starting onday 0. Each limb was evaluated and scored on a 0 to 4 scale every 3^(rd)day. Results are expressed as the mean arthritic score per limb (maximumscore of 4). Groups of 4 mice per condition were used.

FIG. 13. Therapeutic treatment with anti-α1 mAb can decrease arthriticscore. Wild-type Balb/c mice were treated with anti-collagen mAbs at day0, followed by LPS on day 3. Arthritis was apparent by day 6 andcontinued for several weeks. Mice were treated i.p. with mAbs (250 ug)or Ig fusion protein (200 ug) every 3^(rd) day starting on day 4. Micereceived either mAb (Ha4/8 isotype control or Ha31/8 anti-α1), Ig fusionprotein (Isotype control Ig or TNF-R55-Ig) or a combination of both (250ug Ha31/8 and 200 ug TNF-R55-Ig). Each limb was evaluated and scored ona 0 to 4 scale every 3^(rd) day. Results are expressed as the meanarthritic score per limb (maximum score of 4). Groups of 4 mice percondition were used.

FIG. 14. Location of the Epitope for the anti-α1 I domain Blocking mAbs.FIG. 14A illustrates the amino acid sequence of the rat (top) (SEQ IDNO:5) and human (below) (SEQ ID NO:6) α1-I integrin polypeptidesequences. The residues that comprise the MIDAS (metal ion dependentadhesion site) motif are shown in bold. The human amino acids thatreplaced the corresponding rat residues (RΔH) are shown below the ratsequence in the boxed region. For clarity, residue numbering in the textrefers to this figure. B. Increasing concentrations of mAb AJH10 werebound to plates coated with 30 μg/ml human (circles), rat (triangles) orRΔH (squares) α1-I domain. Data shown is representative of threeexperiments.

FIG. 15. FIG. 15 illustrates the amino acid sequence of the human α1-Iintegrin polypeptide sequence. The amino acid sequence of the epitopefor the anti-α1-I domain blocking mAbs (SEQ ID NO:8) is shown in thebox.

FIG. 16. Identification of a blocking mAb to the α1-I domain. FIG. 16Ais a graph showing increasing concentration of mAbs AEF3 (triangles) orAJH10 (circles) were bound to plates coated with 30 μg/ml α1-I domain.FIG. 16B is a graph showing the effect on α1-I domain with increasingconcentrations of mAb AJH10 (diamonds) or mAb BGC5 (squares) and boundcollagen IV (2 μg/ml) coated plates. FIG. 16C is a graph showing theeffect increasing concentrations of mAbs AEF3 (triangles) or AJH10(circles) on K562-α1 cells binding to collagen IV (5 μg/ml) coatedplates. 45-50% of cells added to each well adhered to collagen IV. Datashown is representative of three independent experiments.

FIG. 17. Species Cross-reactivity of the blocking mAbs. FIG. 17A depictsdetergent lysates from (1) sheep vascular smooth muscle, (2) humanleukemia K562-α1 cells or (3) purified RΔH GST-I domain; (4) Rat GST-α1I domain; and (5) human GST-α1 I domain separated by 10-20% SDS-PAGEunder non-reducing conditions, and immunoblotted with function-blockingmAb AJH10. Molecular weight markers are shown on the left; non-reducedα1β1 integrin migrates at ˜180 kDa; GST-I domain migrates at ˜45 kDa.FIG. 17B shows results from rabbit vascular smooth muscle cellsincubated with either mAb AJH10 (bottom) or murine IgG control (top) andanalyzed by fluorescence activated cell sorter (FACS).

FIG. 18. The α1-I domain binds collagen. FIG. 18A is a graph showingthat increasing concentrations of the human α1-I domain were bound toplates previously coated with 1 μg/ml collagen I (squares) or collagenIV (circles). Values shown have been corrected for background binding toBSA. FIG. 18B is a graph showing that 2 μg/ml human α1-I domain wasmixed with increasing concentration of an anti-human α1 integrinantibody 5E8D9 (squares) or an anti-human α2-integrin antibody A2IIE10(circles), and then bound to plates previously coated with 1 μg/mlcollagen IV. FIG. 18C is a graph showing results from plates coated with1 μg/ml collagen IV or 3% BSA. α1-I domain (2 μg/ml) was subsequentlybound to the coated plates in the presence of 1 mM Mn²⁺, 1 mM Mg²⁺, or 5mM EDTA. Data shown is representative of three independent experiments.

DETAILED DESCRIPTION OF THE INVENTION

It is a discovery of the present invention that an antibody to anintegrin and fragment thereof, particularly, an α1-integrin subunit, canblock the interaction of pro-inflammatory leukocytes with components ofthe extracellular matrix including, but not limited to collagens,laminin and fibronectin. While not intending to limit the invention toany single mechanism of action it is proposed that disruption of theinteraction between the integrin and fragment thereof and thesurrounding matrix may decrease the expression of pro-inflammatorycytokines. It is further proposed that antibodies to integrins andfragments thereof may be modulating the effector phases of inflammatoryresponses by acting at the level of the antigen-specific T cell. Inaddition, it is proposed that antibodies to integrins and fragmentsthereof may act by disrupting cell migration within tissues and/oreffects on cellular priming and activation within tissues.

This discovery illustrates the importance of adhesion molecules of theintegrin family, particularly α1β1, in the peripheral tissue environmentduring conditions related to inflammation. It also extends the role ofintegrins family and fragments thereof in inflammation beyond leukocyteattachment and extravasation at the endothelial interface byhighlighting the importance of the matrix-rich peripheral tissueenvironment to immune responses and it reveals peripheral tissues as anew point of intervention for adhesion based therapies.

The methods of the present invention contemplate the use of antibodiesto integrins where the integrins contemplated include molecules whichcomprise a β chain, including but not limited to β1, β2, β3, β4, β5, β6,β7, β8, non-covalently bound to an α chain, including but not limited toα1, α2, α3, α4, α5, α6, α7, α8, α9, α10, αV, αL, αM, αX, αD, αE, αIIb.Examples of the various integrins contemplated for use in the inventioninclude, but are not limited to:

-   -   α1β1, α2β1, α3β1, α4β1, ═5β1, α6β1, α7β1, α8β1, α9β1, α10β1,        αVβ1, αLβ1, αMβ1, αXβ1, αDβ1, αI I bβ1, αEβ1;    -   α1β2, α2β2, α3β2, α4β2, α5β2, α6β2, α7β2, α8β2, α9β2, α10β2,        αVβ2, αLβ2, αMβ2, αXβ2, αDβ2, αI I bβ2, αEβ2;    -   α1β3, α2β3, α3β3, α4β3, α5β3, α6β3, α7β3, α8β3, α9β3, α10β3,        αVβ3, αLβ3, αMβ3, αXβ3, αDβ3, αI I bβ3, αEβ3;    -   α1β4, α2β4, α3β4, α4β4, α5β4, α6β4, α7β4, α8β4, α9β4, α10β4,        αVβ4, αLβ4, αMβ4, αXβ4 αDβ4, αI I bβ4, αEβ4;    -   α1β5, α2β5, α3β5, α4β5, α5β5, α6β5, α7β5, α8β5, α9β5, α10β5,        αVβ5, αLβ5, αMβ5, αXβ5, αDβ5, αI I bβ5, αEβ5;    -   α1β6, α2β6, α3β6, α4β6, α5β6, α6β6, α7β6, α8β6, α9β6, α10β6,        αVβ6, αLβ6, αMβ6, αXβ6, αDβ6, αI I bβ6, αEβ6;    -   α1β7, α2β7, α3β7, α4β7, α5β7, α6β7, α7β7, α8β7, α9β7, α10β7,        αVβ7, αLβ7, αMβ7, αXβ7, αDβ7, αI I bβ7, αEβ7;    -   α1β8, α2β8, α3β8, α4β8, α5β8, α6β8, α7β8, α8β8, α9β8, α10β8,        αVβ8, αLβ8, αMβ8, αXβ8, αDβ8, αI I bβ8, αEβ8;

The methods of the present invention also contemplate the use ofantibodies to integrin fragments including for example antibodies to a βchain alone, including but not limited to β1, β2, β3, β4, β5, β6, β7,β8, as well as an cc chain alone, including but not limited to α1, α2,α3, α4, α5, α6, α7, α8, α9, α10, αV, αL, αM, αX, αD, αE, αIIb. Inaddition, the methods of the present invention further contemplate theuse of antibodies to integrin fragments including for example antibodiesto the I domain of the α chain, including but not limited to the Idomain from α1β1 (Briesewitz et al., 1993 J. Biol. Chem. 268:2989); α2β1(Takada and Hemler, 1989 J Cell Biol 109:397), αLβ2 (Larson et al., 1989J Cell Biol 108:703), αMβ2 (Corbi et al., 1988 J Biol Chem 263:12403),αXβ2 (Corbi et al., 1987 EMBO J 6:4023), αDβ2 (Grayson et al., 1988 JExp Med 188:2187), αEβ7 (Shaw et al., 1994 J Biol Chem 269:6016). In apreferred embodiment, the α1-I domain antigenic determinant comprises anamino acid sequence of at least 6 contiguous amino acids, wherein thecontiguous sequence is found within the sequence of FIG. 15. Moreover,in a preferred embodiment, the contiguous sequence isVal-Gln-Arg-Gly-Gly-Arg.

Methods for producing integrins for use in the present invention areknown to those of skill in the art (see for e.g. Springer et al. 1990,Nature 346:425-434).

Embodiments of the present invention further include anti-integrinpolyclonal and monoclonal antibodies. Preferred embodiments of thepresent invention include a monoclonal antibody such ananti-α1monoclonal antibody.

An α1β1function blocking antibody as used herein refers to an antibodythat binds to the α1-I domain, specifically at an epitope identified byamino acids 91-96 of FIG. 15, and that blocks α1β1function as tested by,for example, the ability to inhibit K562-α1 dependent adhesion toCollagen IV (see Example 15).

Preferred antibodies and homologs for treatment, in particular for humantreatment, include human antibody homologs, humanized antibody homologs,chimeric antibody homologs, Fab, Fab′, F(ab′)2 and F(v) antibodyfragments, and monomers or dimers of antibody heavy or light chains ormixtures thereof. Thus, monoclonal antibodies against an integrinmolecule and fragment thereof are the preferred binding agent in themethod of the invention.

As used herein, the term “antibody homolog” includes intact antibodiesconsisting of immunoglobulin light and heavy chains linked via disulfidebonds. The term “antibody homolog” is also intended to encompass aprotein comprising one or more polypeptides selected from immunoglobulinlight chains, immunoglobulin heavy chains and antigen-binding fragmentsthereof which are capable of binding to one or more antigens (i.e., α1,α2, α6 or alpha-I domain containing integrin subunits). The componentpolypeptides of an antibody homolog composed of more than onepolypeptide may optionally be disulfide-bound or otherwise covalentlycrosslinked.

Accordingly, therefore, “antibody homologs” include intactimmunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypesthereof), wherein the light chains of the immunoglobulin may be of typeskappa or lambda.

“Antibody homologs” also include portions of intact antibodies thatretain antigen-binding specificity, for example, Fab fragments, Fab′fragments, F(ab′)2 fragments, F(v) fragments, heavy chain monomers ordimers, light chain monomers or dimers, dimers consisting of one heavyand one light chain, and the like. Thus, antigen-binding fragments, aswell as full-length dimeric or trimeric polypeptides derived from theabove-described antibodies are themselves useful.

As used herein, a “humanized antibody homolog” is an antibody homolog,produced by recombinant DNA technology, in which some or all of theamino acids of a human immunoglobulin light or heavy chain that are notrequired for antigen binding have been substituted for the correspondingamino acids from a nonhuman mammalian immunoglobulin light or heavychain.

As used herein, a “chimeric antibody homolog” is an antibody homolog,produced by recombinant DNA technology, in which all or part of thehinge and constant regions of an immunoglobulin light chain, heavychain, or both, have been substituted for the corresponding regions fromanother immunoglobulin light chain or heavy chain. In another aspect theinvention features a variant of a chimeric molecule which includes: (1)an integrin targeting moiety; (2) optionally, a second peptide, e.g.,one which increases solubility or in vivo life time of the integrintargeting moiety, e.g., a member of the immunoglobulin super family orfragment or portion thereof, e.g., a portion or a fragment of IgG, e.g.,the human IgG1 heavy chain constant region, e.g., CH2 and CH3 hingeregions; and a toxin moiety. The chimeric molecule can be used to treata subject, e.g., a human, at risk for disorder related to proliferationof epithelial cells such as hair follicles and the like.

As used herein, a “human antibody homolog” is an antibody homologproduced by recombinant DNA technology, in which all of the amino acidsof an immunoglobulin light or heavy chain that are derived from a humansource.

As used herein, “an inflammatory disorder”, includes, but is not limitedto, such disorders as, skin related conditions such as psoriasis,eczema, burns and dermatitis. Other inflammatory disorders contemplatedfor treatment by the methods of the present invention include but arenot limited to the treatment of asthma, bronchitis, menstrual cramps,tendinitis, bursitis, and the treatment of pain and headaches, or as anantipyretic for the treatment of fever. The methods of the inventionalso would be useful to treat gastrointestinal conditions such asinflammatory bowel disease, Crohn's disease, gastritis, irritable bowelsyndrome and ulcerative colitis and for the prevention of colorectalcancer. The methods of the invention would be useful in treatinginflammatory disorders in such diseases as vascular diseases, migraineheadaches, periarteritis nodosa, thyroiditis, aplastic anemia, Hodgkin'sdisease, rheumatic fever, type I diabetes, myasthenia gravis, multiplesclerosis, sarcoidosis, nephrotic syndrome, Behcet's syndrome,polymyositis, gingivitis, hypersensitivity, conjunctivitis, swellingoccurring after injury, myocardial ischemia, and the like. The methodsof the invention are also useful in the treatment of allergic rhinitis,respiratory distress syndrome, endotoxin shock syndrome, andatherosclerosis.

In a preferred embodiment, the methods of the invention are useful inthe treatment of arthritis, including for example, rheumatoid arthritisand osteoarthritis.

An “effective amount” is an amount sufficient to effect beneficial ordesired clinical results. An effective amount can be administered in oneor more administrations. In terms of treatment of an inflammatorydisorder, an “effective amount” of an anti-integrin antibody is anamount sufficient to palliate, ameliorate, stabilize, reverse, slow ordelay progression of an inflammation-related condition in accordancewith clinically acceptable standards for disorders to be treated or forcosmetic purposes. Detection and measurement of indicators of efficacymay be measured by a number of available diagnostic tools, including butnot limited to, for example, by physical examination including bloodtests, pulmonary function tests, and chest X-rays; CT scan;bronchoscopy; bronchoalveolar lavage; lung biopsy and CT scan.

The technology for producing monoclonal antibodies, including forexample, anti-integrin monoclonal antibodies is well known. See forexample, Mendrick et al. 1995, Lab. Invest. 72:367-375 (mAbs to murineanti-α1β1 and anti-α2β1); Sonnenberg et al. 1987 J. Biol. Chem.262:10376-10383 (mAbs to murine anti-α6β1); Yao et al. 1996, J Cell Sci1996 109:3139-50 (mAbs to murine anti-α7β1); Hemler et al. 1984, JImmunol 132:3011-8 (mAbs to human α1β1); Pischel et al. 1987 J Immunol138:226-33 (mAbs to human α2β1); Wayner et al. 1988, J Cell Biol107:1881-91 (mAbs to human α3β1); Hemler et al. 1987 J Biol Chem262:11478-85 (mAbs to human α4β1); Wayner et al. 1988 J Cell Biol107:1881-91 (mAbs to human α5β1); Sonnenberg et al. 1987, J. Biol. Chem.262:10376-10383 (mAbs to human α6β1); A Wang et al. 1996 Am. J. Respir.Cell Mol. Biol. 15:664-672 (mAbs to human α9β1); Davies et al. 1989 JCell Biol 109:1817-26 (mAbs to human αV β1); Sanchez-Madrid et al. 1982,Proc Natl Acad Sci USA 79:7489-93 (mAbs to human αL β2); Diamond et al.1993, J Cell Biol 120:1031-43 (mAbs to human αMβ2); Stacker et al. 1991J Immunol 146:648-55 (mAbs to human αXβ2); Van der Vieren et al 1995Immunity 3:683-90 (mAbs to human αDβ2); Bennett et al. 1983 Proc NatlAcad Sci USA 80:2417-21 (mAbs to human αI I bβ3); Hessle et al. 1984,Differentiation 26:49-54 (mAbs to human α6β4); Weinacker et al. 1994 JBiol Chem 269:6940-8 (mAbs to human αVβ5); Weinacker et al. 1994 J BiolChem 269:6940-8 (mAbs to human αVβ6); Cerf-Bensussan et al 1992 Eur JImmunol 22:273-7 (mAbs to human αEβ7); Nishimura et al. 1994 J Biol Chem269:28708-15 (mAbs to human αVβ8); Bossy et al. 1991 EMBO J 10:2375-85(polyclonal antisera to human α8β1); Camper et al. 1998 J. Biol. Chem.273:20383-20389 (polyclonal antisera to human α10β1).

In general, an immortal cell line (typically myeloma cells) is fused tolymphocytes (typically splenocytes) from a mammal immunized with wholecells expressing a given antigen, e.g., an integrin, and the culturesupernatants of the resulting hybridoma cells are screened forantibodies against the antigen. See, generally, Kohler et at., 1975,Nature 265: 295-497, “Continuous Cultures of Fused Cells SecretingAntibody of Predefined Specificity”.

Immunization may be accomplished using standard procedures. The unitdose and immunization regimen depend on the species of mammal immunized,its immune status, the body weight of the mammal, etc. Typically, theimmunized mammals are bled and the serum from each blood sample isassayed for particular antibodies using appropriate screening assays.For example, anti-integrin antibodies may be identified byimmunoprecipitation of 125I-labeled cell lysates fromintegrin-expressing cells. Antibodies, including for example,anti-integrin antibodies, may also be identified by flow cytometry,e.g., by measuring fluorescent staining of antibody-expressing cellsincubated with an antibody believed to recognize integrin molecules. Thelymphocytes used in the production of hybridoma cells typically areisolated from immunized mammals whose sera have already tested positivefor the presence of anti-integrin antibodies using such screeningassays.

Typically, the immortal cell line (e.g., a myeloma cell line) is derivedfrom the same mammalian species as the lymphocytes. Preferred immortalcell lines are mouse myeloma cell lines that are sensitive to culturemedium containing hypoxanthine, arninopterin and thymidine (“HATmedium”). Typically, HAT-sensitive mouse myeloma cells are fused tomouse splenocytes using 1500 molecular weight polyethylene glycol (“PEG1500”). Hybridoma cells resulting from the fusion are then selectedusing HAT medium, which kills unfused and unproductively fused myelomacells (unfused splenocytes die after several days because they are nottransformed). Hybridomas producing a desired antibody are detected byscreening the hybridoma culture supernatants. Is For example, hybridomasprepared to produce anti-integrin antibodies may be screened by testingthe hybridoma culture supernatant for secreted antibodies having theability to bind to a recombinant integrin-expressing cell line.

To produce antibody homologs, including for example, anti-integrinantibody homologs, that are intact immunoglobulins, hybridoma cells thattested positive in such screening assays were cultured in a nutrientmedium under conditions and for a time sufficient to allow the hybridomacells to secrete the monoclonal antibodies into the culture medium.Tissue culture techniques and culture media suitable for hybridoma cellsare well known. The conditioned hybridoma culture supernatant may becollected and the anti-integrin antibodies optionally further purifiedby well-known methods.

Alternatively, the desired antibody may be produced by injecting thehybridoma cells into the peritoneal cavity of an unimmunized mouse. Thehybridoma cells proliferate in the peritoneal cavity, secreting theantibody which accumulates as ascites fluid. The antibody may beharvested by withdrawing the ascites fluid from the peritoneal cavitywith a syringe.

Fully human monoclonal antibody homologs against, for example integrins,are another preferred binding agent which may block antigens in themethod of the invention. In their intact form these may be preparedusing in vitro-primed human splenocytes, as described by Boerner et al.,1991, J. Immunol. 147:86-95, “Production of Antigen-specific HumanMonoclonal Antibodies from In Vitro-Primed Human Splenocytes”.

Alternatively, they may be prepared by repertoire cloning as describedby Persson et al., 1991, Proc. Nat. Acad. Sci. USA 88: 2432-2436,“Generation of diverse high-affinity human monoclonal antibodies byrepertoire cloning” and Huang and Stollar, 1991, J. Immunol. Methods141: 227-236, “Construction of representative immunoglobulin variableregion CDNA libraries from human peripheral blood lymphocytes without invitro stimulation”. U.S. Pat. No. 5,798,230 (Aug. 25, 1998, “Process forthe preparation of human monoclonal antibodies and their use”) describespreparation of human monoclonal antibodies from human B cells. Accordingto this process, human antibody-producing B cells are immortalized byinfection with an Epstein-Barr virus, or a derivative thereof, thatexpresses Epstein-Barr virus nuclear antigen 2 (EBNA2). EBNA2 function,which is required for immortalization, is subsequently shut off, whichresults in an increase in antibody production.

In yet another method for producing fully human antibodies, U.S. Pat.No. 5,789,650 (Aug. 4, 1998, “Transgenic non-human animals for producingheterologous antibodies”) describes transgenic non-human animals capableof producing heterologous antibodies and transgenic non-human animalshaving inactivated endogenous immunoglobulin genes. Endogenousimmunoglobulin genes are suppressed by antisense polynucleotides and/orby antiserum directed against endogenous immunoglobulins. Heterologousantibodies are encoded by immunoglobulin genes not normally found in thegenome of that species of non-human animal. One or more transgenescontaining sequences of unrearranged heterologous human immunoglobulinheavy chains are introduced into a non-human animal thereby forming atransgenic animal capable of functionally rearranging transgenicimmunoglobulin sequences and producing a repertoire of antibodies ofvarious isotypes encoded by human immunoglobulin genes. Suchheterologous human antibodies are produced in B-cells which arethereafter immortalized, e.g., by fusing with an immortalizing cell linesuch as a myeloma or by manipulating such B-cells by other techniques toperpetuate a cell line capable of producing a monoclonal heterologous,fully human antibody homolog.

Yet another preferred binding agent which may block integrin antigens orfragments thereof in the method of the invention is a humanized antibodyhomolog having the capability of binding to an integrin protein orfragments thereof. Following the early methods for the preparation ofchimeric antibodies, a new approach was described in EP 0239400 (Winteret al.) whereby antibodies are altered by substitution of theircomplementarity determining regions (CDRs) for one species with thosefrom another. This process may be used, for example, to substitute theCDRs from human heavy and light chain Ig variable region domains withalternative CDRs from murine variable region domains. These altered Igvariable regions may subsequently be combined with human Ig constantregions to created antibodies which are totally human in compositionexcept for the substituted murine CDRs. Such CDR-substituted antibodieswould be predicted to be less likely to elicit an immune response inhumans compared to chimeric antibodies because the CDR-substitutedantibodies contain considerably less non-human components. The processfor humanizing monoclonal antibodies via CDR “grafting” has been termed“reshaping”. (Riechmann et al., 1988 Nature 332: 323-327, “Reshapinghuman antibodies for therapy”; Verhoeyen et al., 1988, Science 239:1534-1536, “Reshaping of human antibodies using CDR-grafting inMonoclonal Antibodies”.

Typically, complementarity determining regions (CDRs) of a murineantibody are transplanted onto the corresponding regions in a humanantibody, since it is the CDRs (three in antibody heavy chains, three inlight chains) that are the regions of the mouse antibody which bind to aspecific antigen. Transplantation of CDRs is achieved by geneticengineering whereby CDR DNA sequences are determined by cloning ofmurine heavy and light chain variable (V) region gene segments, and arethen transferred to corresponding human V regions by site directedmutagenesis. In the final stage of the process, human constant regiongene segments of the desired isotype (usually gamma I for CH and kappafor CL) are added and the humanized heavy and light chain genes areco-expressed in mammalian cells to produce soluble humanized antibody.

The transfer of these CDRs to a human antibody confers on this antibodythe antigen binding properties of the original murine antibody. The sixCDRs in the murine antibody are mounted structurally on a V region“framework” region. The reason that CDR-grafting is successful is thatframework regions between mouse and human antibodies may have verysimilar 3-D structures with similar points of attachment for CDRS, suchthat CDRs can be interchanged. Such humanized antibody homologs may beprepared, as exemplified in Jones et al., 1986 Nature 321: 522-525,“Replacing the complementarity-determining regions in a human antibodywith those from a mouse”; Riechmann, 1988, Nature 332:323-327,“Reshaping human antibodies for therapy”; Queen et al., 1989, Proc. Nat.Acad. Sci. USA 86:10029, “A humanized antibody that binds to theinterleukin 2 receptor” and Orlandi et al., 1989, Proc. Natl. Acad. Sci.USA 86:3833 “Cloning Immunoglobulin variable domains for expression bythe polymerase chain reaction”.

Nonetheless, certain amino acids within framework regions are thought tointeract with CDRs and to influence overall antigen binding affinity.The direct transfer of CDRs from a murine antibody to produce ahumanized antibody without any modifications of the human V regionframeworks often results in a partial or complete loss of bindingaffinity. In a number of cases, it appears to be critical to alterresidues in the framework regions of the acceptor antibody in order toobtain binding activity.

Queen et al., 1989, Proc. Nat. Acad. Sci. USA 86: 10029-10033, “Ahumanized antibody that binds to the interleukin 2 receptor” and WO90/07861 (Protein Design Labs Inc.) have described the preparation of ahumanized antibody that contains modified residues in the frameworkregions of the acceptor antibody by combining the CDRs of a murine mAb(anti-Tac) with human immunoglobulin framework and constant regions.They have demonstrated one solution to the problem of the loss ofbinding affinity that often results from direct CDR transfer without anymodifications of the human V region framework residues; their solutioninvolves two key steps. First, the human V framework regions are chosenby computer analysts for optimal protein sequence homology to the Vregion framework of the original murine antibody, in this case, theanti-Tac MAb. In the second step, the tertiary structure of the murine Vregion is modeled by computer in order to visualize framework amino acidresidues which are likely to interact with the murine CDRs and thesemurine amino acid residues are then superimposed on the homologous humanframework. Their approach of employing homologous human frameworks withputative murine contact residues resulted in humanized antibodies withsimilar binding affinities to the original murine antibody with respectto antibodies specific for the interleukin 2 receptor (Queen et al.,1989 [supra]) and also for antibodies specific for herpes simplex virus(HSV) (Co. et al., 1991, Proc. Nat. Acad. Sci. USA 88: 2869-2873,“Humanized antibodies for antiviral therapy”.

According to the above described two step approach in WO 90/07861, Queenet al. outlined several criteria for designing humanizedimmunoglobulins. The first criterion is to use as the human acceptor theframework from a particular human immunoglobulin that is usuallyhomologous to the non-human donor immunoglobulin to be humanized, or touse a consensus framework from many human antibodies. The secondcriterion is to use the donor amino acid rather than the acceptor if thehuman acceptor residue is unusual and the donor residue is typical forhuman sequences at a specific residue of the framework. The thirdcriterion is to use the donor framework amino acid residue rather thanthe acceptor at positions immediately adjacent to the CDRS

One may use a different approach (see Tempest, 1991, Biotechnology 9:266-271, “Reshaping a human monoclonal antibody to inhibit humanrespiratory syncytial virus infection in vivo”) and utilize, asstandard, the V region frameworks derived from NEWM and REI heavy andlight chains respectively for CDR-grafting without radical introductionof mouse residues. An advantage of using the Tempest et al., 1991approach to construct NEWM and REI based humanized antibodies is thatthe 3dimensional structures of NEWM and REI variable regions are knownfrom x-ray crystallography and thus specific interactions between CDRsand V region framework residues can be modeled.

The subject treatments are effective on both human and animal subjectsafflicted with these conditions. Animal subjects to which the inventionis applicable extend to both domestic animals and livestock, raisedeither as pets or for commercial purposes. Examples are dogs, cats,cattle, horses, sheep, hogs and goats.

In the methods of the invention the antibodies, including for example,anti-VLA-1 antibody may be administered parenterally. The term“parenteral” as used herein includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional and intracranial injection orinfusion techniques.

The pharmaceutical compositions of this invention comprise any of thecompounds of the present invention, or pharmaceutically acceptablederivatives thereof, together with any pharmaceutically acceptablecarrier. The term “carrier” as used herein includes known acceptableadjuvants and vehicles.

According to this invention, the pharmaceutical compositions may be inthe form of a sterile injectable preparation, for example a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to techniques known in the art using suitabledispersing or wetting agents and suspending agents.

The pharmaceutical compositions of this invention may be given orally.If given orally, they can be administered in any orally acceptabledosage form including, but not limited to, capsules, tablets, aqueoussuspensions or solutions.

For topical applications, the pharmaceutical compositions may beformulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers.

The pharmaceutical compositions of this invention may also beadministered by nasal aerosol or inhalation through the use of anebulizer, a dry powder inhaler or a metered dose inhaler.

The dosage and dose rate of the compounds of this invention effective toproduce the desired effects will depend on a variety of factors, such asthe nature of the inhibitor, the size of the subject, the goal of thetreatment, the nature of the pathology to be treated, the specificpharmaceutical composition used, and the judgment of the treatingphysician. Dosage levels of between about 0.001 and about 100 mg/kg bodyweight per day, preferably between about 0.1 and about 50 mg/kg bodyweight per day of the active ingredient compound are useful. Mostpreferably, the antibody homologs will be administered at a dose rangingbetween about 0.1 mg/kg body weight/day and about 20 mg/kg bodyweight/day, preferably ranging between about 0.1 mg/kg body weight/dayand about 10 mg/kg body weight/day and at intervals of every 1-14 days.In another preferred embodiment the antibody is administered at a doseof about 0.3 to 1 mg/kg when administered I.P. In another preferredembodiment, the antibody is administered at a dose of about 5 to 12.5mg/kg when administered I.V. Preferably, an antibody composition isadministered in an amount effective to provide a plasma level ofantibody of at least 1 ug/ml.

Persons having ordinary skill in the art can readily test if anantagonist of the invention is having it intended effect. For instance,cells contained in a sample of the individual's epithelium are probedfor the presence of the agent in vitro (or ex vivo) using a secondreagent to detect the administered agent. For example, this may be afluorochrome labelled antibody specific for the administered agent whichis then measured by standard FACS (fluorescence activated cell sorter)analysis. Alternatively, presence of the administered agent is detectedin vitro (or ex vivo) by the inability or decreased ability of theindividual's cells to bind the same agent which has been itself labelled(e.g., by a fluorochrome). The preferred dosage should producedetectable coating of the vast majority of hedgehog-positive cells.Preferably, coating is sustained in the case of an antibody homolog fora 1-14 day period.

Practice of the present invention will employ, unless indicatedotherwise, conventional techniques of cell biology, cell culture,molecular biology, microbiology, recombinant DNA, protein chemistry, andimmunology, which are within the skill of the art. Such techniques aredescribed in the literature. See, for example, Molecular Cloning: ALaboratory Manual, 2nd edition. (Sambrook, Fritsch and Maniatis, eds.),Cold Spring Harbor Laboratory Press, 1989; DNA Cloning, Volumes I and II(D. N. Glover, ed), 1985; Oligonucleotide Synthesis, (M. J. Gait, ed.),1984; U.S. Pat. No. 4,683,195 (Mullis et al.,); Nucleic AcidHybridization (B. D. Hames and S. J. Higgins, eds.), 1984; Transcriptionand Translation (B. D. Hames and S. J. Higgins, eds.), 1984; Culture ofAnimal Cells (R. I. Freshney, ed). Alan R. Liss, Inc., 1987; ImmobilizedCells and Enzymes, IRL Press, 1986; A Practical Guide to MolecularCloning (B. Perbal), 1984; Methods in Enzymology, Volumes 154 and 155(Wu et al., eds), Academic Press, New York; Gene Transfer Vectors forMammalian Cells (J. H. Miller and M. P. Calos, eds.), 1987, Cold SpringHarbor Laboratory; Immunochemical Methods in Cell and Molecular Biology(Mayer and Walker, eds.), Academic Press, London, 1987; Handbook ofExperiment Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds.), 1986; Manipulating the Mouse Embryo, Cold Spring HarborLaboratory Press, 1986.

The following Examples are provided to illustrate the present invention,and should not be construed as limiting thereof.

Examples Chemical Reagents

Fluorescein isothiocyanate (FITC) was purchased from Sigma Chemical Co.(St. Louis, Mo.). Croton oil was purchased from ICN Biochemicals(Aurora, Ohio). Whole sheep blood in Alsevers solution was obtained fromEast Acres Biologicals (Southbridge, Mass.). Type I rat tail collagenand type IV mouse collagen were purchased from Collaborative ResearchInc. (Bedford, Mass.) and Gibco (Gaithersburg, Md.), respectively.

Balb/c female mice of 6-8 weeks of age were purchased from Taconic(Germantown, N.Y.) and the α1β1 integrin-deficient mice on a Balb/cbackground were as previously described (3).

Example 1

Monoclonal Antibodies. Function-blocking mAbs to murine antigens wereprepared in an azide-free and low endotoxin format: Ha31/8 (hamsteranti-CD49a; integrin α1) (Mendrick et al. 1995. Lab. Invest.72:367-375), Ha1/29 (hamster anti-CD49b; integrin α2) (β1) (Mendrick etal. 1995. Lab. Invest. 72:367-375; Mendrick, D. L. and D. M. Kelly 1993Lab. Invest. 69:690-702), hamster group II control mAb Ha4/8 (hamsteranti-KLH) (Mendrick, D. L. and D. M. Kelly 1993 Lab. Invest.69:690-702), and PS/2 (rat anti-CD49d; integrin α4β1 chain) (Miyake etal. 1991 J. Exp. Med. 173:599-607). In addition, the followingfunction-blocking mAbs to murine antigens were purchased as no-azide/lowendotoxin preparations from Pharmingen (San Diego, Calif.): HMβ1-1(hamster anti-CD29; integrin β1chain) (Noto et al. 1995 Int. Immunol.7:835-842), Ha2/5 (hamster anti-CD29; integrin β1chain) (Mendrick, D. L.and D. M. Kelly 1993 Lab. Invest. 69:690-702), 3E2 (hamster anti-CD54,ICAM-1) (Scheynius et al. 1993 J. Immunol. 150:655-663), 5H10-27 (ratanti-CD49e; integrin α5) (Kinashi, T., and T. A. Springer. 1994. BloodCells. 20:25-44), GoH3 (rat anti-CD49f; integrin α6) (Sonnenberg et al.1987 J. Biol. Chem. 262:10376-10383), and the rat isotype control mAbsR35-95 (rat IgG2a) and R35-38 (rat IgG2b).

Adhesion Assay. Splenocytes from Balb/c mice were cultured with 20 ng/mlIL-2 for 7-12 d. Adhesion of cells to type I and type IV collagen was aspreviously described (Gotwals et al. 1996 J. Clin. Invest.97:2469-2477). Briefly, 96-well Maxisorp plates (Nunc, Napierville,Ill.) were coated with either 10 μg/ml type IV or 5 μg/ml type Icollagen and non-specific sites blocked with 1% BSA. IL-2 activatedsplenocytes were labeled with 2 μM BCECF[2′,7′-bis(carboxyethyl)-5(6)carboxyl fluorescein pentaacetoxymethylester] (Molecular Probes, Eugene, Oreg.) and incubated with10 μg/ml of indicated mAbs for 15 min. 10⁵ cells in 0.25% BSA in RPMIwere then added to coated wells and incubated for 60 min at 37° C.Unbound cells were removed by washing three times with 0.25% BSA inRPMI. Adhesion was quantified using a CytoFluor 2350 fluorescent platereader (Millipore, Bedford, Mass.). The ratio of bound cells to inputcells was measured and percent adhesion relative to control mAb-treatedcells (normalized to 100%) calculated. Background values due to celladhesion on wells coated with BSA alone were subtracted.

Expression and functional blockade of α1β1 and α2β1 on activatedleukocytes. Given the key role leukocytes play in inflammation, wedecided to test whether anti-α1 and anti-α2 mAbs were capable ofblocking leukocyte adhesion to collagens. In order to obtain leukocytesexpressing high levels of both α1 and α2, murine T cells were stimulatedin vitro with IL-2 for 7-12 d. These cells expressed high levels of bothα1 and α2 (FIG. 1A), and bound well to both collagen type IV and typeI-coated surfaces (FIG. 1B). Adhesion to type IV collagen was partiallyinhibited by anti-α1 mAb alone and was not inhibited by anti-α2 mAbalone. In contrast, adhesion to type I collagen was completely inhibitedby anti-α2 mAb and anti-α1 mAb alone showed only partial inhibition.Both anti-β1 mAb and the combination of anti-α1 and anti-α2 mAbscompletely inhibited adhesion to types I and IV collagen. Havingdemonstrated that the α1β1 and α2β1 integrins are expressed on activatedT cells and that anti-α1 and α2 mAbs are able to functionally blockleukocyte adhesion to collagens, we used these mAbs to investigate thein vivo role of these integrins in animal models of inflammatorydisorders.

Example 2

Inhibition of DTH responses by anti-integrin mAbs. SRBC-induced delayedtype hypersensitivity (DTH) responses were adapted from a previouslypublished protocol (Hurtrel et al. 1992 Cell. Immunol. 142:252-263).Briefly, mice were immunized s.c. in the back with 2×10⁷ SRBC in 100 ulPBS on d 0. The mice were challenged on d 5 by injecting 1×10⁸ SRBC in25 ul PBS s.c into the right hind footpad. Footpad thickness wasmeasured with an engineer's caliper (Mitutoyo/MTI, Paramus, N.J.) 20 hafter antigen challenge, and the degree of footpad swelling calculated.Results are reported as the mean percent increase footpad thickness±SEMand calculated as % increase=[1−(Right footpad thickness 20 h afterantigen challenge/Uninjected left footpad thickness 20 h after antigenchallenge)]×100. To block the effector phase of the SRBC-induced DTHresponse, therapeutic or control mAb (100 ug), which were preparedaccording to the methods described in Example 1, was given i.p. 1 hprior to antigen challenge on d 5.

SRBC-induced DTH is a well characterized in vivo model of inflammation,and in particular psoriasis, that has been used to demonstrate theimportance of a variety of cytokines and adhesion molecules ininflammation (Tedder et al. 1995 J. Exp. Med. 181:2259-2264, Terashitaet al. 1996 J. Immunol. 156:4638-4643). SRBC-sensitized mice receivedanti-integrin mAbs 1 h prior to footpad antigen challenge andinflammation was assessed 20 h later as measured by increased footpadthickness. PBS and control hamster Ig-treated mice showed a 60-70%increase in footpad thickness 20 h after antigen challenge (FIG. 2).Compared to control hamster Ig treatment, anti-α1 or anti-α2 mAbsresulted in a 68% and 60% inhibition in footpad thickness, respectively.The combination of anti-α1 and α2 mAbs resulted in 71% inhibition,demonstrating little additive effect over anti-α1 or anti-α2 mAbs alone.Treatment with other anti-integrin mAbs was also effective at inhibitingDTH effector response. The degree of inhibition seen with the variousmAb treatments was 49% (anti-α4), 23% (anti-α5), and 57% (anti-α6).Lastly, mAb blockade of the common β1 integrin subunit (mAb HMBI-1)inhibited the effector DTH response by 67%.

Example 3

Inhibition of CHS effector responses by anti-integrin mAbs. Contacthypersensitivity (CHS) to FITC was assayed as previously described(Gaspari et al. 1991 In Current Protocols in Immunology. J. E. Coligan,A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober,editors. John Wiley & Sons, New York. Section 4.2:1). Briefly, mice weresensitized by painting 100 ul 0.5% FITC in 1:1 acetone/dibutylphthalateonto the shaved back on d 0. 10 d later, animals were challenged byapplying 5 ul 0.5% FITC onto both sides of each ear. Ear swellingresponse was determined by ear thickness measured with an engineer'scaliper (Mitutoyo/MTI, Paramus, N.J.) at the time of antigen challenge(d 10) and 24 h later, and the results reported as mean percent increasein baseline ear thickness±SEM. Increase in ear thickness was calculatedas % increase=[1−(Ear thickness 24 h after antigen challenge/Earthickness at the time of antigen challenge)]×100. To block the effectorphase of the CHS response, therapeutic or control mAb (250 ug) was giveni.p. 4 h prior to antigen challenge on d 10. Mice that wereantigen-sensitized and ear challenged with vehicle only (vehiclecontrol) or mice that were ear challenged without prior sensitization(irritant control) served as negative controls (never exceeded 2%increase in ear thickness).

Given that CHS is mechanistically distinct from DTH and involvesdifferent effector cells, we investigated what effect anti-integrin mAbshad on the effector phase of the CHS response. Mice werehapten-sensitized using FITC applied to their shaved backs, followed 10d later with FITC challenge to the ear resulting in an inflammatoryresponse the next day. FITC-sensitized mice demonstrated a 60-70%increase in thickness 24 h after antigen challenge (FIG. 3). Consistentwith published results (Scheynius et al. J. Immunol. 150:655-663),anti-ICAM-1 mAb treatment resulted in 51% inhibition of ear swelling.Compared to control hamster mAb, treatment of mice with anti-α1 oranti-α2 mAb 4 h prior to antigen challenge resulted in 37% and 57%inhibition in ear swelling, respectively (FIG. 3). The combination ofanti-α1 and anti-α2 mAbs resulted in slightly greater inhibition of earswelling (65%). Treatment with other mAbs to β1 integrins revealed thatwhile anti-α4 and anti-α5 mAbs resulted in no inhibition of FITC-inducedCHS effector response when compared to control rat mAb, treatment withanti-α6 mAb resulted in an 86% inhibition of effector responses. Lastly,mAb blockade of the common β1 integrin subunit inhibited CHS effectorresponses by 74%. Similar CHS results were obtained using differentstrains of mice (C57/BL6, 129/Sv) and a different sensitizing agent(oxazolone) (data not shown). Similar to the results seen in theSRBC-induced DTH model, histologic analysis of inflamed ears revealedthat both edema formation and leukocytic infiltration were inhibited byanti-α1 and anti-α2 mAb treatment.

Consistent with the finding that α1β1 and α2β1 can be expressed onIL-2-activated splenocytes, analysis of lymph nodes fromantigen-sensitized mice (FITC or oxazolone) revealed α1β1 and α2β1 to beexpressed exclusively on CD44^(hi) LFA-1^(hi) activated CD4+ and CD8+ Tcells (data not shown). Treatment of mice with anti-α1 and anti-α2 mAbsdid not result in deletion of these cells, as the numbers of activated Tcells in both spleen and lymph nodes seen in response to antigensensitization in the CHS model was unaffected. In addition, effectorcells were not functionally deleted as prolonged treatment ofantigen-sensitized mice with anti-α1 and anti-α2 mAbs (d 10-16) did notaffect the inflammatory response of mice challenged with antigen at d 20(data not shown).

Example 4

CHS effector responses are decreased in α1β1-deficient mice. To excludethe possibility that the inhibitory role of α1β1 in the effectorresponse of FITC-mediated CHS was mAb-mediated, experiments were carriedout in wild-type and α1β1-integrin deficient mice (FIG. 4). MAbinhibition of the effector phase in wild-type mice was consistent withprevious results, with 56% inhibition in ear thickness seen withanti-α1, 56% with anti-α2, and 62% with a combination of anti-α1 andanti-α2. The effector phase of CHS was significantly reduced inuntreated α1β1-deficient mice as compared to untreated wild-type mice(30% vs 71% increase in ear thickness, respectively). As expected, thelevel of ear swelling in untreated α1β1-deficient mice was equivalent tothe level of ear swelling seen in anti-α1 mAb-treated wild-type mice.Lastly, mAb blockade of α2β1 in the α1β1-deficient mice resulted in onlyslightly increased inhibition of ear swelling, consistent with theresults seen in wild-type mice treated with a combination of anti-α1 andanti-α2 mAbs.

Example 5

To further exclude the possibility that the inhibitory effect of theanti-integrin mAbs seen in both the DTH and CHS models of inflammationis caused by a general anti-inflammatory effect mediated by the anti-α1and anti-α2 mAbs, the effect of these mAbs on irritant dermatitis wasstudied.

To assess irritant dermatitis, mice were painted with 5 ul of 0.8%croton oil in acetone on both sides of each ear. Therapeutic or controlantibodies were given 4 h prior to the application of the irritant. Earswelling was measured 24 h later as described above and compared to earthickness prior to croton oil application. Results are reported as meanpercent increase in baseline ear thickness±SEM as described above. Micepainted with acetone only (vehicle control) served as a negativecontrol.

24 h later, ears of mice treated with croton oil showed a significantincrease in ear thickness (48%), when compared to mice receiving vehicleonly (acetone). Toxic ear swelling caused by croton oil was notsignificantly affected in mice pretreated with anti-α1 or anti-α2 mAbswhen compared to either PBS or control mAb-treated animals (FIG. 5).Histologic examination of the croton oil-treated ears revealed nodifferences in numbers or types of infiltrating cells or edema formationin mice treated with anti-α1 or anti-α2 mAbs, as compared to controlmAb-treated mice or PBS-treated mice (data not shown).

Example 6

Inhibition of arthritis by α1β1 and α2β1. As α1β1 is well expressed oninfiltrating cells in the synovium of arthritis patients, we decided toexamine whether anti-α1 or anti-α2 mAbs would be inhibitory in anaccelerated model of arthritis previously described (Terato et al. 1992J. Immunol. 148:2103-2108; Terato et al. 1995 Autoimmunity. 22:137-147).

Arthrogen-CIA Antibody kits were purchased from Stratagene (La Jolla,Calif.) and arthritis induced using a well established protocol (Teratoet al. 1992 J. Immunol. 148:2103-2108; Terato et al. 1995 Autoimmunity.22:137-147). Briefly, arthritis was induced through i.p. injection of acocktail of 4 anti-collagen type II mAbs (1 mg each) on d 0, followed byi.p. injection of 50 ug LPS on d 3. Over the course of the next 3-4 d,the mice developed swollen wrists, ankles and digits. Therapeutic orcontrol mAb (250 ug) was administered i.p. 4 h prior to injection of theanti-collagen mAbs on d 0, and again 4 h prior to LPS administration ond 3, and then continuing every 3^(rd) day for the length of theexperiment. Beginning on d 3, mice were evaluated for the development ofarthritis. Severity of arthritis in each limb was scored using a fourpoint system. 0=normal; 1=mild redness, slight swelling of ankle orwrist; 2=moderate swelling of ankle or wrist; 3=severe swellingincluding some digits, ankle, and foot; 4=maximally inflamed.

Severe arthritis in Balb/c mice developed within 72 h after LPSinjection and persisted for more than 3 weeks. Neither injection ofanti-collagen mAbs alone nor LPS alone induced arthritis. Mice receivingcontrol mAb treatment displayed equally severe arthritis as than seen inPBS-treated mice (FIG. 6). In contrast, treatment with anti-α1 mAb aloneresulted in a marked reduction (78%) in arthritis, lasting the durationof the experiment. Treatment with anti-α2 mAb alone also had abeneficial effect, resulting in a 32% decrease in the arthritic score ascompared to control mAb-treated mice. The combination of anti-α1 andanti-α2 mAbs resulted in a similar degree of inhibition as seen withanti-α1 mAb alone.

Example 7

Histological analysis of effect of anti-α1 and anti-α2 mAb treatment onthe inflammatory cellular infiltrate. Further histological analysis ofthe SRBC-induced DTH response confirmed the ability of anti-α1 andanti-α2 mAb treatment to modulate the elicited inflammatory response(FIG. 7). An unchallenged footpad from an SRBC-sensitized mouse (FIG. 7Panel A) showed virtually no inflammatory cellular infiltrate whencompared to an SRBC-challenged footpad from the same mouse (FIG. 7 PanelB). Treatment of SRBC-sensitized mice with anti-α1 and anti-α2 mAbseither alone or combined greatly reduced the number of theseinfiltrating cells found in SRBC-challenged footpads when compared tocontrol mAb-treated mice (FIG. 7 Panel C-F). Closer examination of theinfiltrating cells revealed most cells to be composed of neutrophils,with some monocytes and lymphocytes present, and confirmed that anti-α1and anti-α2 mAb treatment greatly decreased the numbers of these cells(FIG. 7 Panel G-H).

Example 8

Immunohistochemical demonstration of α1-expressing cells in theinflammatory cellular infiltrate. Immunohistochemistry was performed tomore precisely determine the nature of the infiltrating cells andwhether they express collagen-binding integrins (FIG. 8). Infiltratingcells from an inflamed footpad of an untreated mouse were examined forexpression of α1β1 integrin and cell lineage markers (FIG. 8). α1 β1integrin was found to be expressed on many infiltrating leukocytes (FIG.8A). Dual immunohistochemistry was utilized to identify the nature ofthe infiltrating cells and the distribution of α1 β1 expression (FIG.8B). Using cell lineage markers, the infiltrate was found to be composedlargely of granulocyte/monocytes (Mac-1+), with many of these cellsbeing neutrophils (Gr1+), along with a smaller number of T lymphocytes(CD3+) (FIG. 8B). Expression of α1 β1 integrin was found among all threesubsets of cells, with α1 expressed on a subset of Mac-1+granulocyte/monocytes, a subset of Gr1+ neutrophils, and on the majorityof infiltrating CD3+ T lymphocytes (FIG. 8B). Detailedimmunohistochemical analysis revealed that although anti-α1 and anti-α2mAb treatment reduced the numbers of infiltrating cells, no change inthe cellular composition of the infiltrate was seen (data not shown).Immunohistochemistry staining with a FITC anti-hamster mAb confirmed theability of the anti-α1 and anti-α2 mAb to localize to the inflamedfootpad (data not shown).

Example 9

Inhibition of arthritis by mAbs to α1β1 and α2β1 and in α1-deficientmice. As α1β1 is well expressed on infiltrating cells in the synovium ofarthritis patients, we decided to examine whether anti-α1 or anti-α2mAbs would be inhibitory in an accelerated model of arthritis previouslydescribed (Terato et al. 1992 J. Immunol 148:2103-2108; Terato et al.1995 Autoimmunity 22:137-147). This model involves injection of acocktail of anti-collagen type II mAbs into mice, followed later by LPSadministration, resulting in the development of arthritis over the next3-7 d. Mice were given mAb every 3^(rd) day starting at d 0, and scoredfor the development of arthritis every 3^(rd) day. Severe arthritisdeveloped in all mice within 72 h after LPS injection and persisted formore than 3 weeks. Neither injection of anti-collagen mAbs alone nor LPSalone induced arthritis. Mice receiving control mAb treatment displayedequally severe arthritis as than seen in PBS-treated mice (FIG. 9A). Incontrast, treatment with anti-α1 mAb alone resulted in a markedreduction (79% and higher) in arthritis, lasting the duration of theexperiment. Treatment with anti-α2 mAb alone also had a beneficialeffect, resulting in a 37% decrease in the arthritic score as comparedto control mAb-treated mice. The combination of anti-α1 and anti-α2 mAbsresulted in a similar degree of inhibition as seen with anti-α1 mAbalone. Reduction of arthritic score with anti-α1 mAb treatment was seenin all mice and compares favorably with several other mAb-basedtreatments for arthritis such as soluble TNF receptor Ig fusion protein(Mori et al. 1996 J. Immunol. 157:3178-3182), anti-Mac-1 (Taylor et al.1996 Immunology. 88:315-321), anti-α4 (Seiffge 1996 J. Rheumatol.23:2086-2091), and anti-ICAM-1 (Kakimoto et al. 1992 Cell Immunol.142:326-337) (FIG. 9A). In agreement with mAb-based data showing animportant role for α1β1 in arthritis, untreated α1-deficient mice showedsignificant reduction in arthritic score when compared to wild-type mice(FIG. 9B).

Example 10

Effect of anti-α1 mAb treatment on the immunopathology of arthriticjoints. Joints from wild-type arthritic mice (day 8) receiving eithercontrol mAb or anti-α1 mAb treatment were compared visually andhistologically to joints from a normal untreated mouse (FIG. 10).Visually, joints from control mAb-treated mice demonstrated redness andswelling of the entire foot including digits, while anti-α1 mAb-treatedmice showed little if any signs of inflammation in either joints ordigits. Histologic examination showed severe changes in controlmAb-treated arthritic joints, with extensive infiltration of thesubsynovial tissue with inflammatory cells, adherence of cells to thejoint surface, and marked cartilage destruction as evidenced byproteoglycan loss (FIG. 10). Consistent with previous reports (Terato etal. 1992 J. Immunol 148:2103-2108; Terato et al. 1995 Autoimmunity22:137-147), the majority of the infiltrating cells in this model areneutrophils. Anti-α1 mAb treatment of mice dramatically reduced theamount of inflammatory infiltrate and the degree of cartilagedestruction (FIG. 10).

Example 11

Development of arthritis is delayed in the absence of lymphocytes andinhibition of arthritis by anti-α1 mAb occurs in the absence oflymphocytes. To determine what cell types might be important in thecollagen mAb-induced arthritis model we compared the ability ofwild-type B6-129 mice and RAG-1-deficient B6-129 mice to developarthritis (FIG. 11). Genetic deletion of the RAG-1 (recombinationactivating gene-1) gene results in a complete loss of mature T and Blymphocytes (Mombaerts et al. 1992 Cell 68:869-877). Both the wild-typeand RAG-1-deficient mice developed arthritis, though the kinetics ofinduction in the RAG-1-deficient mice is significantly slower (FIG. 11).These results suggest that while lymphocytes are involved in this modelof arthritis, they are not required for the development and progressionof the disease. Published reports examining the effect of theRAG-1-deficient mice in other models of arthritis also found that lossof T and B lymphocytes delayed the onset of arthritis (Plows et al. 1999J. Immunol. 162:1018-1023). Treatment of either wild-type orRAG-1-deficient mice with anti-α1 mAb completely inhibited arthritis(FIG. 11). These results demonstrate that the effectiveness of anti-α1mAb in this model is not dependent on the presence of lymphocytes, andthat as suggested by previous experiments (FIG. 9), the efficacy ofanti-α1 mAb in preventing disease may be through its action on otherα1-expressing cells, such as macrophages and neutrophils.

Example 12

Dose response of anti-α1 mAb inhibition of arthritis. Given the strikingeffects of anti-α1 mAb treatment on preventing arthritis, we extendedthese studies to include a dose response analysis (FIG. 12). Differentdoses of mAb were administered i.p. every 3^(rd) day starting at day 0.In agreement with earlier data, a 250 ug dose of anti-α1 mAb resulted innear complete prevention of arthritis. A lower dose of 100 ug of anti-α1mAb was partially effective at preventing arthritis in this model, whilelower doses did not have any discernable effect on arthritic score (FIG.12).

Example 13

Therapeutic treatment with anti-α1 mAb can decrease arthritic score.Given the effectiveness of anti-α1 mAb in preventing arthritis, weattempted to treat mice that are on their way to develop disease.Arthritis was induced in mice by injection of a cocktail ofanti-collagen type II mAbs on day 0, followed by LPS administration onday 3. Mice were then treated with either anti-α1 mAb or a soluble TNFreceptor Ig fusion protein starting on day 4. Progression of arthritiswas completely blocked in mice receiving anti-α1 mAb starting at day 4,when compared to mice receiving control hamster mAb starting at day 4(FIG. 13). The degree of inhibition seen with therapeutic administrationof anti-α1 mAb was complete and was equal to that seen with preventativetreatment of anti-α1 mAb (started at day 0) (FIG. 13). In comparison,treatment with TNF receptor Ig fusion protein from day 4 onwardsresulted in only a 60-70% inhibition in arthritic score when compared tocontrol Ig fusion protein (FIG. 13). Combined treatment of anti-α1 mAband TNF receptor Ig fusion together was effective at completelyinhibiting arthritic score, which is not surprising given the completeeffectiveness of anti-α1 mAb treatment alone in suppressing arthritis.In summary, these results indicate that therapeutic treatment withanti-α1 mAb is effective at inhibiting arthritic score, and comparesfavorably to therapeutic treatment with a TNF antagonist.

Example 14

Cloning and mutagenesis of the α1-I domain. Human and rat α1β1 integrinI domain sequences were amplified from full length cDNAs (Kern, et al.(1994) J. Biol. Chem. 269, 22811-22816; Ignatius et al. (1990) J. CellBiol. 111, 709-720) by the polymerase chain reaction (PCR) (PCR COREKit; Boehringer Mannheim, GmbH Germany), using either human specific(5′-CAGGATCCGTCAGCCCCACATTTCAA-3′ [forward] (SEQ ID NO:1);5′-TCCTCGAGGGCTTGCAGGGCAAATAT-3′ [reverse] (SEQ ID NO:2)) or ratspecific (5′-CAGGATCCGTCAGTCCTACATTTCAA-3′ [forward] (SEQ ID NO:3);5′-TCCTCGAGCGCTTCCAAAGCGAATAT-3′ [reverse] (SEQ ID NO:4)) primers. Theresulting PCR amplified products were purified, ligated into pGEX4t-i(Pharmacia), and transformed into competent DH5α cells (LifeTechnologies). Ampicillin resistant colonies were screened for theexpression of the ˜45 kDa glutathione S-transferase-I domain fusionprotein. The sequences from inserts of plasmid DNA of clones that wereselected for further characterization were confirmed by DNA sequencing.

A rat/human chimeric α1-I domain (RΔH) was generated (MORPH Mutagenesiskit; 5 prime-3 prime), exchanging the rat residues G91, R92, Q93, andL96 (FIG. 14A) for the corresponding human residues, V, Q, R, and R,respectively. Clones harboring the RΔH I domain were identified by theloss of a diagnostic Stu 1 restriction enzyme site, and the insertsconfirmed by DNA sequencing. The amino acid sequence of the human α1-Idomain is shown in FIG. 15.

Example 15

Generation of mAbs specific to the -α1 I domain. Monoclonal antibodieshave proved to be very useful probes in studying the relationshipbetween structure and function of integrin subunits. For example, mAbswere used extensively to study regions of the β1 subunit associated withan activated conformation (Qu, A., and Leahy, D. J. (1996) Structure 4,931-942). Thus, to identify potential probes for conformational changesof the α1-I domain, we generated a panel of mAbs to the human α1-Idomain.

Generation of anti-α1 I domain Monoclonal Antibodies. FemaleRobertsonian mice (Jackson Labs) were immunized intraperitoneally (i.p.)with 25 μg of purified human α1β1 (Edwards et al. (1995) J. Biol. Chem.270, 12635-12640) emulsified with complete Fruend's adjuvant(LifeTechnologies). They were boosted three times i.p. with 25 μg ofα1β1 emulsified with incomplete Freunds's adjuvant (LifeTechnologies).The mouse with the highest anti-α1-I domain titer was boosted i.p. with100 μg of α1β1 three days prior to fusion, and intravenously with 50 μgof α1β1 one day prior to fusion. Spleen cells were fused with FL653myeloma cells at a 1:6 ratio and were plated at 100,000 and 33,000 perwell into 96 well tissue culture plates.

Supernatants were assessed for binding to the α1β1 integrin by singlecolor FACS. Prior to FACS analysis, supernatants were incubated withuntransfected K562 cells to eliminate IgG that bound solely to the βsubunit. Subsequently, 3-5×10⁴ K562 cells transfected with the α1integrin subunit (K562-α1) suspended in FACS buffer (1% fetal calf serum(FCS) in PBS containing 0.5% NaN₃) were incubated with supernatant for45 minutes at 4° C., washed and incubated with anti-mouse IgG conjugatedto phycoerythrin. After washing twice with FACS buffer, cells wereanalyzed in a Becton Dickinson Flow Cytometer.

Supernantants from the resulting hybridomas were screened for binding tothe α1-I domain. Briefly, 50 μl of 30 μg/ml human α1-I domain-GST fusionin PBS was coated onto wells of a 96-well plate (Nunc) overnight at 4°C. The plates were washed with PBS, blocked with 1% BSA in PBS and thehybridoma supernatant was incubated with the I domain at roomtemperature for 1 hour. After extensive washing with PBS containing0.03% Tween 20, alkaline phosphatase linked anti-mouse IgG (JacksonImmunoResearch) was added for an additional hour. After a final wash, 1mg/ml p-nitrophenylphosphate (pNPP) in 0.1 M glycine, 1 mM ZnCl₂, and 1mM MgCl₂ was added for 30 minutes at room temperature, and the plateswere read at O.D. 405.

Selected supernatants were tested for their ability to inhibit K562-α1dependent adhesion to Collagen IV. K562-α1 cells were labeled with 2 mM2′,7′ (bis-2-carboxyethyl-5 and 6) carboxyfluorescein pentaacetoxymethylester (BCECF; Molecular Probes) in DMEM containing 0.25%BSA at 37° C. for 30 minutes. Labeled cells were washed with bindingbuffer (10 mM Hepes, pH 7.4; 0.9% NaCl; and 2% glucose) and resuspendedin binding buffer plus 5 mM MgCl₂ at a final concentration of 1×10⁶cells/ml. 50 μl of supernatant was incubated with an equal volume of2×10⁵ K562-α1 cells in wells of a 96 well plate. The plate was thencentrifuged and the supernatants removed. Cells were resuspended inbinding buffer and transferred to wells of a collagen-coated plate andincubated for 1 hour at 37° C. Following incubation, the non-adherentcells were removed by washing three times with binding buffer. Attachedcells were analyzed on a Cytofluor (Millipore).

We initially identified 19 hybridomas, the supernatants of which boundto human leukemia K562 cells expressing the α1β1 integrin (K562-α1) andto the α1-I domain. The immunoglobulins were purified from each of thesehybridomas and tested for the ability to block either K562-α1 or α1-Idomain binding to collagen IV. The mAbs fall into two classes: thosethat block and those that do not block α1β1 function. For example, whilethe mAbs produced by clones AEF3, BGC5 and AJH10 bind the α1-I domain(FIG. 16A, data not shown for BGC5), only mAb AJH10 inhibits α1-Idomain-dependent (FIG. 16B) or K562-α1 (FIG. 16C) adhesion to collagenIV. The hybridoma that produces the α-domain antibody AJH10 wasdeposited under the Budapest Treaty on Aug. 2, 2001, with the AmericanType Culture Collection, 10801 University Boulevard, Manassas, Va.20110-2209 (ATCC PTA-3580). Other materials necessary to make AJH10 areavailable to the public domain to those of ordinary skill in the art.

Sequencing of the Complementarity Determining Regions. To establish theclonal origin of this panel of mAbs, we amplified by PCR and sequencedthe CDRs from 12 of the 19 antibodies (data not shown).

2 μg of mRNA, isolated from 10⁷ hybridomas (FastTrack mRNA isolationkit, Invitrogen), was reverse transcribed (Ready-To-Go You Prime FirstStrand Kit, Pharmacia Biotech) using 25 pM each of the followingprimers: heavy chain VH1FOR-2 (Michishita et al. (1993) Cell 72,857-867); light chain, VK4FOR, which defines four separate oligos (Kernet al. (1994) J. Biol. Chem. 269, 22811-22816). For each hybridoma,heavy and light chains were amplified in four separate PCR reactionsusing various combination of the following oligos: 1) Heavy chain:VH1FR1K (Kamata et al. (1995) J. of Biol. Chem. 270, 12531-12535),VH1BACK, VH1BACK (Baldwin et al.(1998) Structure 6, 923-935), V_(H)fr1a,V_(H)fr1b, V_(H)fr1e, V_(H)fr1f, V_(H)fr1g (Ignatius et al. (1990) J.Cell Biol. 111, 709-720), or VH1FOR-2 (Michishita, M., Videm, V., andArnaout, M. A. (1993) Cell 72, 857-867); 2) Light chain: VK1BACK(Baldwin et al. (1998) Structure 6, 923-935), VK4FOR, VK2BACK oligos(Kern et al. (1994) J. Biol. Chem. 269, 22811-22816), or V_(K)fr1a,V_(H)fr1c, V_(H)fr1e, V_(H)fr1f (Ignatius et al. (1990) J. Cell Biol.111, 709-720). Products were amplified (5 min at 95° C., 50 cycles of 1min at 94° C., 2 min at 55° C., 2 min at 72° C., and a final cycle of 10min at 72° C.), gel purified (QIAquick, Qiagen), and sequenced directlyusing various of the listed oligos on an ABI 377 Sequencer.

Sequences from clones producing function-blocking mAbs were nearlyidentical across all the complementarity-determining regions (CDRs) andthe intervening framework regions suggesting that these hybridomas areclonally related.

Example 16

Immunoblotting and FACS Analysis. Sequences of the variable regions ofthe non-blocking antibodies were markedly different from the clonallyrelated family of sequences found for the blocking antibodies. As theblocking antibodies appear to originate from a single clone, we choseone (AJH10) to characterize further.

Immunoblotting The smooth muscle cell layer dissected from sheep aorta,and K562-α1 cells were extracted with 1% Triton X-100 in 50 mM Hepes, pH7.5, 150 mM NaCl, 10 mM phenylmethylsulfonyl flouride (PMSF), 20 μg/mlaprotinin, 10 μg/ml leupeptin, 10 mM ethylenediaminetetraacetic acid(EDTA). Samples were subjected to 4-20% gradient SDS-PAGE, andelectroblotted onto nitrocellulose membranes. The blots were blockedwith 5% dry milk in TBS; washed in TBS containing 0.03% Tween-20, andincubated with antibodies in blocking buffer containing 0.05% NaN₃ for 2hours. Blots were then washed as before, incubated with horseradishperoxidase conjugated anti-mouse IgG for one hour, washed again and thentreated with ECL reagent (Amersham). Blots were then exposed to film(Kodak) for 30 to 60 seconds, and developed.

Immunoblotting (FIG. 17A) and FACS analysis (FIG. 17B) demonstrate thatAJH10 reacts with human, rabbit, and sheep, but not rat α1β1 integrinsuggesting that the blocking mAbs bind to an evolutionarily conserved,linear epitope. The non-blocking mAbs were neither efficient atimmunoblotting nor did they react with species other than human.

Example 17

Binding of the α1-I Domain to Collagen is Divalent Cation-Dependent

A. Purification of the α1-I Domains.

The α1-I domains were expressed in E. coli as GST(glutathione-S-transferase) fusion proteins containing a thrombincleavage site at the junction of the sequences. The clarifiedsupernatant from cells lysed in PBS was loaded onto a glutathioneSepharose™ 4B column (Pharmacia) which was washed extensively with PBS.The α1-I domain-GST fusion protein was eluted with 50 mM Tris-HCl, pH8.0, 5 mM glutathione (reduced). For denaturation studies, the I domainwas cleaved with thrombin in 50 mM Tris, pH 7.5, and purified from theGST fusion partner. DTT was added to 2 mM and the sample was loaded on aglutathione Sepharose™ 4B column. The flow-through and wash fractionswere pooled and loaded onto a Q Sepharose™ FF column (Pharmacia). Theα1-I domain was eluted with 50 mM Tris HCl, pH 7.5, 10 mM2-mercaptoethanol, 75 mM NaCl. The purified I domain displayed itspredicted mass (Lee et al. (1995) Structure 3, 1333-1340, 871 Da) byelectrospray ionization-mass spectrometry (ESI-MS), migrated as a singleband by SDS-PAGE, and the protein eluted as a single peak of appropriatesize by size exclusion chromatography on a Superose™ 6 FPLC column(Pharmacia).

B. Functional Analysis

96 well plates were coated overnight at 4° C. with 1 μg/ml collagen IV(Sigma) or collagen Type I (Collaborative Biomedical), washed withTriton buffer (0.1% Triton X-100; 1 mM MnCl₂; 25 mM Tris-HCl; 150 mMNaCl), and blocked with 3% bovine serum albumin (BSA) in 25 mM Tris-HCl;150 mM NaCl (TBS). Serial dilutions of the α1-I domain-GST fusionprotein in TBS containing 1 mM MnCl₂ and 3% BSA were incubated on thecoated plates at room temperature for 1 hour, and washed in Tritonbuffer. Bound α1-I domain was detected with serial additions of 10 μg/mlbiotinylated anti-GST polyclonal antibody (Pharmacia);ExtrAvidin-horseradish peroxidase (Sigma) diluted 1:3000 in TBScontaining 1 mM MnCl₂ and 3% BSA, and 1-Step ABTS(2,2′-Azine-di[3-ethylbenzthiazoline sulfonate]; Pierce). Plates wereread at O.D. 405 on a microplate reader (Molecular Devices).

Results.

The human and rat (95% identity to human) α1-I domains were expressed inE. coli as GST-fusion proteins and purified over glutathione Sepharose™.Both proteins were examined for binding to collagen I and IV using avariation of an ELISA-based assay previously described (Qu, A., andLeahy, D. J. (1995) Proc. Natl. Acad. Sci. USA 92, 10277-10281). Thehuman α1-I domain binds collagen IV with better efficiency than collagenI (FIG. 18A). An antibody specific to the α1-I domain, but not anantibody specific to the α2-I domain (FIG. 18B) abrogated binding toboth ligands (data for collagen I is not shown). Both Mn²⁺ and Mg²⁺stimulated binding, and EDTA reduced binding to background levels (FIG.18C). No measurable differences in ligand binding were detected betweenthe human and rat α1-I domains suggesting that the sequence differencesbetween species are not functionally relevant (data not shown). Thus,the α1-I domain, specifically, require cation for efficient ligandbinding.

Example 18

A Cation-Dependent Epitope Resides near the MIDAS motif. We exploitedthe observation that AJH10 recognizes the human, but not the rat α1-Idomain sequences to map the epitope for the α1β1 function-blocking mAbs.The human and rat sequences differ by only 12 amino acids, 4 of whichlie in a stretch of 6 amino acids (aa 91-96, FIG. 14A) adjacent to thecritical glutamine (FIG. 14A, aa 97) within the MIDAS motif. To test thehypothesis that the 6 amino acid residues, Val-Gln-Arg-Gly-Gly-Arg,comprise the epitope for the blocking mAbs, we constructed a chimeric Idomain (RΔH), which exchanged the rat residues G91, R92, Q93, and L96for the corresponding human residues, V, Q, R, and R, respectively.AJH10, along with all the function-blocking mAbs, recognizes thechimeric I domain (RΔH; FIG. 14B).

To orient these residues with respect to the MIDAS domain in thetertiary structure of the α1-I domain, we modeled the α1-I domain usingthe coordinates of the crystal structure of the α2 I domain.

A homology model of the human α1 I-domain was built using the X-raycrystal structure of the human α2 I-domain (Ward et al. (1989) Nature341, 544-546). The model was built using the homology modeling module ofInsight II (version 2.3.5; Biosym Technologies). The program CHARMM(Clackson et al. (1991) Nature 352, 624-628) was used with theall-hydrogen parameter set 22 with a distant dependent dielectricconstant of two times the atom separation distance. We first did 1000steps of steepest descent minimization with mass-weighted harmonicpositional constraints of 1 kcal/(mol Å²) on all atoms of the α1-Idomain. This minimization was followed by another 1000 steps of steepestdescent and 5000 steps of Adopted-Basis Newton Raphson with constraintsof 0.1 kcal/(mol Å²) on the C-α atoms of the α1-I domain to avoidsignificant deviations from the α2-I domain X-ray crystal structure.

The α1β1 and α2β1 integrin sequences exhibit 51% identity with noinsertions or deletions, suggesting that the overall structure of thetwo I domains will be similar. The metal coordination site is predictedto be the same in the α1-I domain as in the α2-I domain, and theresidues that comprise the epitope for the blocking mAbs lie on a loopbetween helix α3 and helix α4 which contains the threonine within theMIDAS motif critical for cation binding. The α1-I domain model predictsthat the amide nitrogen of Q92 (FIG. 14A) hydrogen bonds with thecarbonyl group of I33, the residue adjacent to S32. Thus, the loop thatcontains the epitope may play a functional role in stabilizing the MIDASregion.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications will be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention, which is delineated by the appended claims.

1. A method for treating arthritis comprising administering to a subjecthaving arthritis a composition comprising a function blocking antibody,or fragment of said antibody, wherein said antibody or fragment of saidantibody is capable of binding an epitope of VLA-1, wherein the epitopecomprises amino acid residues Val-Gln-Arg-Gly-Gly-Arg (SEQID NO:8) andwherein the antibody or fragment of said antibody is capable of bindinga human α1-I domain but not to a rat α1-I domain.
 2. A method accordingto claim 1, wherein the antibody or fragment thereof is administered inan amount effective to provide a decrease in arthritic score of about79% or greater.
 3. A method according to claim 1, wherein the antibodyor fragment thereof is administered in an amount effective to provide adecrease in arthritic score of about 85% or greater.
 4. A methodaccording to claim 1, wherein the antibody or fragment thereof isadministered in an amount effective to provide a decrease in arthriticscore of about 90% or greater.
 5. A method according to claim 1, whereinthe antibody is monoclonal.
 6. A method according to claim 1, whereinthe subject is a human.
 7. A method according to claim 1, wherein thesubject has rheumatoid arthritis.
 8. A method according to claim 1,wherein the antibody or fragment thereof is administered in an amounteffective to provide a decrease in arthritic score of about 65% orgreater.
 9. A method according to claim 1, wherein the antibody orfragment thereof is administered by orally, subcutaneously,intravenously, intramuscular, intraarticular, intrasynovial,intrasternal, intrathecal, intrahepatically, intralesionally,intracranially, intraparenterally or intranasally.
 10. A methodaccording to claim 1, wherein the antibody or fragment thereof isadministered to the subject in an amount between about 0.1 mg/kg/day toabout 50 mg/kg/day.
 11. A method according to claim 1, wherein theantibody or fragment thereof is administered to the subject in an amountbetween about 0.1 mg/kg/day to about 20 mg/kg/day.
 12. A methodaccording to claim 1, wherein the antibody or fragment thereof isadministered to the subject in an amount between about 0.1 mg/kg/day toabout 10 mg/kg/day.
 13. A method according to claim 1, wherein theantibody or fragment thereof is administered to the subject in an amountbetween about 0.3 mg/kg/day to about 1 mg/kg/day.
 14. A method accordingto claim 1, wherein the antibody or fragment thereof is administered tothe subject in an amount between about 5 mg/kg/day to about 12.5mg/kg/day.
 15. A method according to claim 1, wherein the antibody orfragment thereof is administered to the subject at intervals of everyone to 14 days.